This invention relates to the 37-kDa Streptococcus pneumoniae surface adhesin A protein. Specifically, the invention relates to an isolated nucleic acid encoding the 37-kDa protein of Streptococcus pneumoniae, to unique fragments of the nucleic acid encoding the 37-kDa protein of Streptococcus pneumoniae, and to the polypeptides encoded by those nucleic acids. The invention further relates to antibodies to those polypeptides, and to methods of detecting the presence of Streptococcus pneumoniae, methods of preventing Streptococcus pneumoniae infection, and methods of treating a Streptococcus pneumoniae infection.
Pneumococcal disease continues to be a leading cause of sickness and death in the United States and throughout the world. Both the lack of efficacy of the currently used polysaccharide vaccines in children under 2 years of age and their variable serotype-specific efficacy among vaccinated individuals, have prompted manufacturers to investigate alternative vaccine formulations that do not require the use of multiple capsular polysaccharides. One current approach under consideration is the use of immunogenic species-common proteins as vaccine candidates. These proteins could be used in combination with other immunogenic proteins or as protein carriers in a protein-polysaccharide or oligosaccharide conjugate vaccine. An effective vaccine that included a common protein could eliminate the need for formulations based on multiple capsular polysaccharides (as in the 23-valent polysaccharide vaccine) by offering a broader range of protection against a greater number of serotypes. Additionally, a protein-based vaccine would be T-cell dependent and provide a memory response, resulting in a more efficacious vaccine.
Of the reported pneumococcal proteins, only pneumolysin and the pneumococcal surface protein A (PspA) have been extensively examined for their suitability as vaccine candidates. While both have been shown to be partially protective in mice (Paton et al. 1983. xe2x80x9cEffect of immunization with pneumolysin on survival time of mice challenged with Streptococcus pneumoniae.xe2x80x9d Infect. Immun. 40:548-552 and McDaniel et al. 1991. xe2x80x9cPspA, a surface protein of Streptococcus Pneumoniae, is capable of eliciting protection against pneumococci of more than one capsular type.xe2x80x9d Infect. Immun. 59:222-228), there are disadvantages to their use as vaccine immunogens. Pneumolysin, although well conserved among pneumococci, has been shown to have strong toxic effects in its native state (AlonsoDeVelasco et al. 1995. xe2x80x9cStreptococcus pneumoniae: Virulence factors, pathogenesis, and vaccines.xe2x80x9d Microbiol. Rev. 59:591-603). Recombinant derivatives of reduced toxicity have been produced, and while they show promise in animal protection studies (Alexander et al. 1994. xe2x80x9cImmunization of mice with pneumolysin toxoid confers a significant degree of protection against at least nine serotypes of Streptococcus pneumoniae. Infect. Immun. 62:5683-5688) the problem of maintaining maximal immunogenicity and eliminating toxicity to humans is still in question. PspA, on the other hand, is serologically and structurally heterogeneous. (Crain et al. 1990. xe2x80x9cPneumococcal surface protein A (PspA) is serologically highly variable and is expressed by all clinically important capsular serotypes of Streptococcus pneumoniae.xe2x80x9d Infect. Immun. 58:3293-3299). Its use in vaccine formulations would require multiple PspA types, thus increasing the complexity of vaccine preparation.
An immunogenic species-common protein has been identified from Streptococcus pneumoniae. (Russell et al. 1990. xe2x80x9cMonoclonal antibody recognizing a species-specific protein from Streptococcus pneumoniae.xe2x80x9d J. Clin. Microbiol. 28:2191-2195 and U.S. Pat. No. 5,422,427 in which the 37-kDa protein is referred to as pneumococcal fimbrial protein A). The 37-kDa S. pneumoniae protein has been the focus of several studies and has been designated pneumococcal surface adhesin protein A (PsaA). Immunoblot analysis studies using anti-PsaA monoclonal antibody showed that PsaA is common to all 23 pneumococcal vaccine serotypes (Russell et al. 1990). Enzyme-linked-immunosorbent assay studies have indicated that patients with pneumococcal disease show an antibody increase in convalescent-phase serum to PsaA compared with acute-phase serum antibody levels (Tharpe et al. 1995. xe2x80x9cPurification and seroreactivity of pneumococcal surface adhesin A (PsaA).xe2x80x9d Clin. Diagn. Lab. Immunol. 3:227-229 and Tharpe et al. 1994. xe2x80x9cThe utility of a recombinant protein in an enzyme immunoassay for antibodies against Streptococcus pneumoniae.xe2x80x9d abstr. V-2, p. 617. 1994. American Society for Microbiology, Washington, D.C.). Additionally, a limited in vivo protection study showed that antibodies to the 37-kDa protein protect mice from lethal challenge (Talkington et al. 1996. xe2x80x9cProtection of mice against fatal pneumococcal challenge by immunization with pneumococcal surface adhesin A (PsaA).xe2x80x9d Microbial Pathogenesis 21:17-22).
The gene encoding PsaA from S. pneumoniae strain R36A (an unencapsulated strain) has been cloned in Escherichia coli and sequenced, but this serotype does not contain a 37kDa protein encoding nucleic acid that is highly conserved among the various serotypes. (Sampson et al. 1994. xe2x80x9cCloning and nucleotide sequence analysis of psaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton protein homologous to previously reported Streptococcus sp. adhesins.xe2x80x9d Infect. Immun. 62:319-324). This particular nucleic acid and polypeptide, therefore, are of limited value for use as diagnostic reagents, in infection prevention, in infection treatment, or in vaccine development.
Sequence conservation is a necessary requirement for a candidate species-common vaccine. At present, there are no studies that have investigated the sequence conservation of the psaA gene among pneumococcal types, specifically among encapsulated pneumococci which cause the vast majority of serious disease. Therefore, a need exists to investigate the conservation of the gene in order to provide a polypeptide which can serve as a vaccine for multiple strains of Streptococcus pneumoniae. The present invention fulfills that need by analyzing psaA genes from the 23 serotypes in the 23-valent polysaccharide vaccine and by providing a polypeptide and antibodies to that polypeptide which are conserved among the S. pnuemoniae serotypes and which confer protection to Streptococcus pneumoniae infection.
In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention, in one aspect, relates to an isolated nucleic acid encoding the 37-kDa protein of Streptococcus pneumoniae as set forth in the Sequence Listing as SEQ ID NO:2. The invention also provides unique fragments of at least 10 nucleotides of the nucleic acid set forth in the Sequence Listing as SEQ ID NO:1, which can be used in methods to detect the presence of Streptococcus pneumoniae in a sample and as immunogenic vaccines.
The invention further provides a purified polypeptide encoded by the nucleic acid encoding the 37-kDa protein of Streptococcus pneumoniae as set forth in the Sequence Listing as SEQ ID NO:1, which can be used as immunogenic vaccines.
In another aspect, the invention provides purified antibodies which bind to the 37-kDa protein of Streptococcus pneumoniae or fragments thereof These antibodies can be used in methods to detect the presence of Streptococcus pneumoniae in a sample and in therapeutic and prophylactic methods.
The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this application pertains.
The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Examples included therein.
Before the present compounds and methods are disclosed and described, it is to be understood that this invention is not limited to specific proteins, specific methods, or specific nucleic acids, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
It must be noted that, as used in the specification and the appended claims, the singular forms xe2x80x9ca,xe2x80x9d xe2x80x9can,xe2x80x9d and xe2x80x9cthexe2x80x9d include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to xe2x80x9ca nucleic acidxe2x80x9d includes multiple copies of the nucleic acid and can also include more than one particular species of nucleic acid molecule.
Nucleic Acids
In one aspect, the invention provides an isolated nucleic acid encoding the 37-kDa protein of Streptococcus pneumoniae as set forth in the Sequence Listing as SEQ ID NO:2. The term xe2x80x9cisolatedxe2x80x9d refers to a nucleic acid which is essentially separated from other genes that naturally occur in S. pneumoniae. In one embodiment, the present invention provides an isolated nucleic acid encoding the 37-kDa protein of Streptococcus pneumoniae wherein the nucleic acid is the nucleic acid set forth in the Sequence Listing as SEQ ID NO:1.
The nucleic acids of the present invention can include the positive and/or negative RNA strand as well as the sense and/or nonsense DNA strand, or any combinations thereof These nucleic acids include the genomic DNA fragment encoding the 37-kDa protein and any subgenomic nucleic acids, including DNA and RNA, in the organism encoding all, or a fragment of the 37-kDa protein. The nucleic acid can also be modified, such as nucleic acids containing methylated bases.
This nucleic acid can comprise the coding sequence for the 37-kDa protein itself, or the coding sequence with the gene""s upstream and downstream regulatory sequences, or any combination thereof This nucleic acid can, for example, comprise a DNA and include its own promoter, or another promoter can be operatively linked to the nucleic acid such that the coding sequence of the 37-kDa protein is expressed. Alternatively, the nucleic acid can comprise an RNA such that the RNA is translated and the resulting polypeptide comprises the 37-kDa protein. Therefore sequences normally found associated with the 37-kDa protein coding sequence, such as the promoter, and the translation signals, can be substituted, deleted, or modified.
An isolated nucleic acid comprising a unique fragment of at least 10 nucleotides of the nucleic acid set forth in the Sequence Listing as SEQ ID NO:1 is also provided. Unique fragments, as used herein means a nucleic acid of at least 10 nucleotides that is not identical to any other known nucleic acid sequence. Examples of the sequences of at least 10 nucleotides that are unique to the nucleic acid set forth in the Sequence Listing as SEQ ID NO:1 can be readily ascertained by comparing the sequence of the nucleic acid in question to sequences catalogued in GenBank, or any other sequence database, using computer programs such as DNASIS (Hitachi Engineering, Inc.) or Word Search or FASTA of the Genetics Computer Group (GCG) (Madison, Wis.), which search the catalogued nucleotide sequences for similarities to the nucleic acid in question. If the sequence does not match any of the known sequences, it is unique. For example, the sequence of nucleotides 1-10 can be used to search the databases for an identical match. If no matches are found, then nucleotides 1-10 represent a unique fragment. Next, the sequence of nucleotides 2-11 can be used to search the databases, then the sequence of nucleotides 3-13, and so on up to nucleotides 1320 to 1330 of the sequence set forth in the Sequence Listing as SEQ ID NO:1. The same type of search can be performed for sequences of 11 nucleotides, 12 nucleotides, 13 nucleotides, etc. The possible fragments range from 10 nucleotides in length to 1 nucleotide less than the sequence set forth in the Sequence Listing as SEQ ID NO:1. These unique nucleic acids, as well as degenerate nucleic acids can be used, for example, as primers for amplifying nucleic acids from other strains of Streptococcus pneumoniae in order to isolate allelic variants of the 37-kDa protein, or as primers for reverse transcription of 37-kDa protein RNA, or as probes for use in detection techniques such as nucleic acid hybridization. One skilled in the art will appreciate that even though a nucleic acid of at least 10 nucleotides is unique to a specific gene, that nucleic acid fragment can still hybridize to many other nucleic acids and therefore be used in techniques such as amplification and nucleic acid detection.
Also provided are nucleic acids which encode alelic variants of the 37-kDa protein of S. pneumoniae set forth in the Sequence Listing as SEQ ID NO:2, and those proteins. As used herein, the term xe2x80x9calelic variationsxe2x80x9d or xe2x80x9calelic variantsxe2x80x9d is used to describe the same, or similar 37-kDa pneumococcal surface adhesin proteins that are diverged from the 37-kDa Streptococcus pneumoniae protein set forth in the Sequence Listing as SEQ ID NO:2 by less than 15% in their corresponding amino acid identity. In another embodiment, these allelic variants are less than 10% divergent in their corresponding amino acid identity. In another embodiment, these allelic variants are less than 7% divergent in their corresponding amino acid identity. In another embodiment, these allelic variants are less than 5% divergent in their corresponding amino acid identity. In another embodiment, these allelic variants are less than 3% divergent in their corresponding amino acid identity. In another embodiment, these allelic variants are less than 2% divergent in their corresponding amino acid identity. In yet another embodiment, these allelic variants are less than 1% divergent in their corresponding amino acid identity. These amino acids can be substitutions within the amino acid sequence set forth in the Sequence Listing as SEQ ID NO:2, they can be deletions from the amino acid sequence set forth in the Sequence Listing as SEQ ID NO:2, and they can be additions to the amino acid sequence set forth in the Sequence Listing as SEQ ID NO:2.
The homology between the protein coding region of the nucleic acid encoding the allelic variant of the 37-kDa protein is preferably less than 20% divergent from the region of the nucleic acid set forth in the Sequence Listing as SEQ ID NO:1 encoding the 37-kDa protein. In another embodiment, the corresponding nucleic acids are less than 15% divergent in their sequence identity. In another embodiment, the corresponding nucleic acids are less than 10% divergent in their sequence identity. In another embodiment, the corresponding nucleic acids are less than 7% divergent in their sequence identity. In another embodiment, the corresponding nucleic acids are less than 5% divergent in their sequence identity. In another embodiment, the corresponding nucleic acids are less than 4% divergent in their sequence identity. In another embodiment, the corresponding nucleic acids are less than 3% divergent in their sequence identity. In another embodiment, the corresponding nucleic acids are less than 2% divergent in their sequence identity. In yet another embodiment, the corresponding nucleic acids are less than 1% divergent in their sequence identity. In particular, the nucleic acid variations can create up to about 15% amino acid sequence variation from the protein set forth in the Sequence Listing as SEQ ID NO:2.
One skilled in the art will appreciate that nucleic acids encoding homologs or allelic variants of the 37-kDa protein set forth in the Sequence Listing as SEQ ID NO:2 can be isolated from related gram-positive bacteria in a manner similar to that used to isolate the nucleic acid set forth in the Sequence Listing of the present invention as SEQ ID NO:1. For example, given the sequence of the primers used to amplify the nucleic acid set forth in the sequence listing as SEQ ID NO:1, one can use these or similar primers to amplify a homologous gene from related gram-positive bacteria.
Alternatively, allelic variants can be identified and isolated by nucleic acid hybridization techniques. Probes selective to the nucleic acid set forth in the Sequence Listing as SEQ ID NO:1 can be synthesized and used to probe nucleic acid from the various serotypes of S. pneumoniae. High sequence complementarity and stringent hybridization conditions can be selected such that the probe selectively hybridizes to allelic variants of the sequence set forth in the Sequence Listing as SEQ ID NO:1. For example, the selectively hybridizing nuicleic acids of the invention can have at least 70%, 80%, 85%, 90%, 95%, 97%, 98% and 99%o complementarity with the segment of the sequence to which it hybridizes. The nucleic acids can be at least 10, 12, 50, 100, 150, 200, 300, 500, 750, or 1000 nucleotides in length. Thus, the nucleic acid can be a coding sequence for the 37-klja protein or fragments thereof that can be used as a probe or primer for detecting the presence of M. tuberculosis. If used as primers, the invention provides compositions including at least two nucleic acids which hybridize with different regions so as to amplify a desired region. Depending on the length of the probe or primer, target region can range between 70% complementary bases and full complementarity and still hybridize under stringent conditions. For example, for the purpose of diagnosing the presence of an allelic variant of the sequence set forth in the Sequence Listing as SEQ ID NO:1, the degree of complementarity between the hybridizing nucleic acid (probe or primer) and the sequence to which it hybridizes is at least enough to distinguish hybridization with a nucleic acid from unrelated bacteria. The invention provides examples of nucleic acids unique to SEQ ID NO:1 in the Sequence Listing so that the degree of complementarity required to distinguish selectively hybridizing from nonselectively hybridizing nucleic acids under stringent conditions can be clearly determined for each nucleic acid. One skilled in the art will appreciate that sequences can be added to either one end or both ends of unique fragments, for example, to aid subsequent cloning, expression, or detection of the fragment.
xe2x80x9cStringent conditionsxe2x80x9d refers to the washing conditions used in a hybridization protocol. In general, the washing conditions should be a combination of temperature and salt concentration chosen so that the denaturation temperature is approximately 5-20xc2x0 C. below the calculated Tm of the nucleic acid hybrid under study. The temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to the probe or protein coding nucleic acid of interest and then washed under conditions of different stringencies. The Tm of such an oligonucleotide can be estimated by allowing 2xc2x0 C. for each A or T nucleotide, and 4xc2x0 C. for each G or C. For example, an 18 nucleotide probe of 50% G+C would, therefore, have an approximate Tm of 54xc2x0 C.
In another aspect, the present invention provides an isolated nucleic acid comprising the nucleic acid as set forth in the Sequence Listing as SEQ ID NO:3.
In another aspect, the present invention provides an isolated nucleic acid comprising the nucleic acid as set forth in the Sequence Listing as SEQ ID NO:4.
The nucleic acid encoding a 37-kDa protein may be obtained by any number of techniques known to one skilled in the art. One method is to synthesize a recombinant nucleic acid molecule. For example, oligonucleotide synthesis procedures are routine in the art and oligonucleotides coding for a particular protein or regulatory region are readily obtainable through automated DNA synthesis. A nucleic acid for one strand of a double-stranded molecule can be synthesized and hybridized to its complementary strand. One can design these oligonucleotides such that the resulting double-stranded molecule has either internal restriction sites or appropriate 5xe2x80x2 or 3xe2x80x2 overhangs at the termini for cloning into an appropriate vector. Double-stranded molecules coding for relatively large proteins or regulatory regions can be synthesized by first constructing several different double-stranded molecules that code for particular regions of the protein or regulatory region, followed by ligating these DNA molecules together. For example, Cunningham et al. (xe2x80x9cReceptor and Antibody Epitopes in Human Growth Hormone Identified by Homolog-Scanning Mutagenesis,xe2x80x9d Science, 243:1330-1336 (1989)), have constructed a synthetic gene encoding the human growth hormone gene by first constructing overlapping and complementary synthetic oligonucleotides and ligating these fragments together. See also, Ferretti, et al. (Proc. Nat. Acad. Sci. 82:599-603 (1986)), wherein synthesis of a 1057 base pair synthetic bovine rhodopsin gene from synthetic oligonucleotides is disclosed. Once the appropriate DNA molecule is synthesized, this DNA can be cloned downstream of a promoter. Techniques such as this are routine in the art and are well documented.
An example of another method of obtaining a nucleic acid encoding a 37-kDa surface adhesin A protein is to isolate that nucleic acid from the organism in which it is found and clone it in an appropriate vector. For example, a DNA or cDNA library can be constructed and screened for the presence of the nucleic acid of interest. The probe used to screen the library can be designed to be selective for the 6B serotype protein. Methods of constructing and screening such libraries are well known in the art and kits for performing the construction and screening steps are commercially available (for example, Stratagene Cloning Systems, La Jolla, Calif.). Once isolated, the nucleic acid can be directly cloned into an appropriate vector, or if necessary, be modified to facilitate the subsequent cloning steps. Such modification steps are routine, an example of which is the addition of oligonucleotide linkers which contain restriction sites to the termini of the nucleic acid. General methods are set forth in Sambrook et al., xe2x80x9cMolecular Cloning, a Laboratory Manual,xe2x80x9d Cold Spring Harbor Laboratory Press (1989).
Yet another example of a method of obtaining a Streptococcal 37-kDa surface adhesin A encoding nucleic acid is to amplify the nucleic acid from the nucleic acids found within the host organism. Amplification procedures are well known to those skilled in the art, for example see Innis et al. xe2x80x9cPCR Protocols: A Guide to Methods and Applicationsxe2x80x9d Academic Press, Inc. 1990. An example of amplification of a nucleic acid encoding the 37-kDa protein of Streptococcus pneumoniae serotype 6B is discussed in the Example contained herein.
37-kDa Protein
The present invention also provides a purified polypeptide as set forth in the Sequence Listing a SEQ ID NO:2 and a purified polypeptide encoded by a nucleic acid comprising a unique fragment of at least 10 nucleotides of SEQ ID NO:1. The protein can be used as a vaccine component as well as a reagent for identifying host antibodies raised against Streptococcus pneumoniae during infection. The purified protein can also be used in methods for detecting the presence of Streptococcus pneumoniae. 
Unique fragments of the 37-kDa protein can be identified in the same manner as that used to identify unique nucleic acids. For example, a sequence of 3 amino acids or more, derived from the sequence of the 37-kDa protein as set forth in the Sequence Listing as SEQ ID NO:2 can be used to search the protein sequence databases. Those that do not match a known sequence are therefore unique.
xe2x80x9cPurified proteinxe2x80x9d as used herein means the protein or fragment is sufficiently free of contaminants or cell components with which the protein normally occurs to distinguish the protein from the contaminants or cell components. It is not contemplated that xe2x80x9cpurifiedxe2x80x9d necessitates having a preparation that is technically totally pure (homogeneous), but purified as used herein means the protein or polypeptide fragment is sufficiently separated from contaminants or cell components with which it normally occurs to provide the protein in a state where it can be used in an assay, such as immunoprecipitation or ELISA. For example, the xe2x80x9cpurifiedxe2x80x9d protein can be in an electrophoretic gel.
Once a nucleic acid encoding a 37-kDa pneumococcal surface adhesin protein of serotype 6B, or a fragment of that nucleic acid, is constructed, modified, or isolated, that nucleic acid can then be cloned into an appropriate vector, which can direct the in vivo or in vitro synthesis of that 37-kDa pneumococcal surface adhesin protein, or fragment thereof The vector is contemplated to have the necessary functional elements that direct and regulate transcription of the inserted gene, or gene fragment. These functional elements include, but are not limited to, a promoter, regions upstream or downstream of the promoter, such as enhancers that may regulate the transcriptional activity of the promoter, an origin of replication, appropriate restriction sites to facilitate cloning of inserts adjacent to the promoter, antibiotic resistance genes or other markers which can serve to select for cells containing the vector or the vector containing the insert, RNA splice junctions, a transcription termination region, or any other region which may serve to facilitate the expression of the inserted gene or gene fragment. (See generally, Sambrook et al.).
There are numerous E. coli (Escherichia coli) expression vectors known to one of ordinary skill in the art which are useful for the expression of the nucleic acid insert. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, a promoter system from phage lambda, or other phage promoters such as T4 or T7 promoters. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences for example, for initiating and completing transcription and translation. If necessary, an amino terminal methionine can be provided by insertion of a Met codon 5xe2x80x2 and in-frame with the downstream nucleic acid insert. Also, the carboxy-terminal extension of the nucleic acid insert can be removed using standard oligonucleotide mutagenesis procedures.
Alternatively, viral expression systems can be used to express the nucleic acid of the present invention, or fragments thereof For example, vaccinia virus vectors can accept large inserts and can be used to express foreign genes for vaccination purposes. (See, e.g., Fredman, T. Science 244:1275 (1989)). Other viral expression systems, such as the baculovirus expression system, are also commonly used in the art.
Additionally, yeast expression can be used. There are several advantages to yeast expression systems. First, evidence exists that proteins produced in a yeast secretion systems exhibit correct disulfide pairing. Second, post-translational glycosylation is efficiently carried out by yeast secretory systems. The Saccharomyces cerevisiae pre-pro-alpha-factor leader region (encoded by the MFxe2x80x3-1 gene) is routinely used to direct protein secretion from yeast. (Brake el al, xe2x80x9cxe2x88x9d-Factor-Directed Synthesis and Secretion of Mature Foreign Proteins in Saccharomyces cerevisiae.xe2x80x9d Proc. Nat. Acad. Sci., 81:4642-4646 (1984)). The leader region of pre-pro-alpha-factor contains a signal peptide and a pro-segment which includes a recognition sequence for a yeast protease encoded by the KEX2 gene: this enzyme cleaves the precursor protein on the carboxyl side of a Lys-Arg dipeptide cleavage signal sequence. The nucleic acid coding sequence can be fused in-frame to the pre-pro-alpha-factor leader region. This construct is then put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter. The nucleic acid coding sequence is followed by a translation termination codon which is followed by transcription termination signals. Alternatively, the nucleic acid coding sequences can be fused to a second protein coding sequence, such as Sj26 or xcex2-galactosidase, used to facilitate purification of the fusion protein by affinity chromatography. The insertion of protease cleavage sites to separate the components of the fusion protein is applicable to constructs used for expression in yeast. Efficient post translational glycosylation and expression of recombinant proteins can also be achieved in Baculovirus systems.
Mammalian cells permit the expression of proteins in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures, addition of lipid moieties, and secretion of active protein. Vectors useful for the expression of active proteins in mammalian cells are characterized by insertion of the protein coding sequence between a strong viral promoter and a polyadenylation signal. The vectors can contain genes conferring hygromycin resistance, gentamicin resistance, or other genes or phenotypes suitable for use as selectable markers, or methotrexate resistance for gene amplification. The chimeric protein coding sequence can be introduced into a Chinese hamster ovary (CHO) cell line using a methotrexate resistance-encoding vector, or other cell lines using suitable selection markers. Presence of the vector DNA in transformed cells can be confirmed by Southern blot analysis. Production of RNA corresponding to the insert coding sequence can be confirmed by Northern blot analysis. A number of other suitable host cell lines capable of secreting intact human proteins have been developed in the art, and include the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cells, etc. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, etc. The vectors containing the nucleic acid segments of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transformation, transduction, and electroporation are commonly utilized for prokaryotic cells, whereas calcium phosphate, DEAE dextran, or lipofectin mediated transfection or electroporation may be used for other cellular hosts.
Alternative vectors for the expression of genes in mammalian cells, those similar to those developed for the expression of human gamma-interferon, tissue plasminogen activator, clotting Factor VIII, hepatitis B virus surface antigen, protease Nexinl, and eosinophil major basic protein, can be employed. Further, the vector can include CMV promoter sequences and a polyadenylation signal available for expression of inserted nucleic acids in mammalian cells (such as COS-7).
Expression of the gene or hybrid gene can be by either in vivo or in vitro. In vivo synthesis comprises transforming prokaryotic or eukaryotic cells that can serve as host cells for the vector. Alternatively, expression of the gene can occur in an in vitro expression system. For example, in vitro transcription systems are commercially available which are routinely used to synthesize relatively large amounts of mRNA. In such in vitro transcription systems, the nucleic acid encoding the 37-kDa pneumococcal surface adhesin protein would be cloned into an expression vector adjacent to a transcription promoter. For example, the Bluescript II cloning and expression vectors contain multiple cloning sites which are flanked by strong prokaryotic transcription promoters. (Stratagene Cloning Systems, La Jolla, Calif.). Kits are available which contain all the necessary reagents for in vitro synthesis of an RNA from a DNA template such as the Bluescript vectors. (Stratagene Cloning Systems, La Jolla, Calif.). RNA produced in vitro by a system such as this can then be translated in vitro to produce the desired 37-kDa pneumococcal surface adhesin protein. (Stratagene Cloning Systems, La Jolla, Calif.).
Another method of producing a 37-kDa pneumococcal surface adhesin protein is to link two peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, Calif.). One skilled in the art can readily appreciate that a peptide or polypeptide corresponding to a 37-kDa pneumococcal surface adhesin protein can be synthesized by standard chemical reactions, either continuous synthesis or step-wise synthesis. For example, a partial polypeptide can be synthesized and not cleaved from its synthesis resin whereas another fragment can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form a 37-kDa pneumococcal surface adhesin protein. (Grant G. A., xe2x80x9cSynthetic Peptides: A User Guide,xe2x80x9d W. H. Freeman and Co., N.Y. (1992) and Bodansky, M and Trost, B., Ed., xe2x80x9cPrinciples of Peptide Synthesis,xe2x80x9d Springer-Verlag Inc., N.Y. (1993)). Alternatively, the 37-kDa pneumococcal surface adhesin protein can by independently synthesized in vivo as described above. Once isolated, these independent polypeptides may be linked to form a 37-kDa pneumococcal surface adhesin protein via similar peptide condensation reactions.
For example, enzymatic ligation of cloned or synthetic peptide segments can allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen et al., Biochemistry, 30:4151 (1991)). Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two step chemical reaction (Dawson et al., xe2x80x9cSynthesis of Proteins by Native Chemical Ligation,xe2x80x9d Science, 266:776-779 (1994)). The first step is the chemoselective reaction of an unprotected synthetic peptide-xe2x88x9d-thioester with another unprotected peptide segment containing an amino-tenninal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site. Application of this native chemical ligation method to the total synthesis of a protein molecule is illustrated by the preparation of human interleukin 8 (IL-8) (Clark-Lewis et al., FEBS Lett., 307:97 (1987), Clark-Lewis et al, J. Biol. Chem., 269:16075 (1994), Clark-Lewis et al., Biochem. 30:3128 (1991), and Rajarathnam et al., Biochem. 29:1689 (1994)).
Alternatively, unprotected peptide segments can be chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer et al., Science, 256:221 (1992)). This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton el al., xe2x80x9cTechniques in Protein Chemistry IV,xe2x80x9d Academic Press, New York, pp. 257-267 (1992)).
The invention also provides fragments of the 37-kDa pneumococcal surface adhesin protein. The polypeptide fragments of the present invention can be recombinant proteins obtained by cloning nucleic acids encoding fragments of the polypeptide in an expression system capable of producing the polypeptide fragments thereof, as described above for the 37-kDa protein. For example, one can determine an immunoreactive region of a 37-kDa pneumococcal surface adhesin protein which can cause a significant immune response, clone the nucleic acid encoding that polypeptide into an expression vector, and isolate that particular polypeptide for further uses, such as diagnostics, therapy, and vaccination. In one example, amino acids found to not contribute to the immunoreactivity and/or specificity can be deleted without a loss in the respective activity.
For example, amino or carboxy-terminal amino acids, can be sequentially removed from the 37-kDa pneumococcal surface adhesin protein and the immunoreactivity tested in one of many available assays. Alternatively, internal amino acids can be sequentially removed and the immunoreactivity tested for each of the deletions. In another example, a fragment of a 37-kDa pneumococcal surface adhesin protein can comprise a modified polypeptide wherein at least one amino acid has been substituted for the naturally occurring amino acid at specific positions, or a portion of either amino terminal or carboxy terminal amino acids, or even an internal region of the polypeptide, can be replaced with a polypeptide fragment or other moiety, such as biotin, which can facilitate in the purification of the modified 37-kDa pneumococcal surface adhesin protein. For example, a modified 37-kDa pneumococcal surface adhesin protein can be fused to a maltose binding protein, through either peptide chemistry of cloning the respective nucleic acids encoding the two polypeptide fragments into an expression vector such that the expression of the coding region results in a hybrid polypeptide. The hybrid polypeptide can be affinity purified by passing it over an amylose affinity column, and the modified 37-kDa pneumococcal surface adhesin protein can then be separated from the maltose binding region by cleaving the hybrid polypeptide with the specific protease factor Xa. (See, e.g., New England Biolabs Product Catalog, 1996, pg. 164.)
Immnunoreactive fragments of a 37-kDa pneumococcal surface adhesin protein can also be synthesized directly or obtained by chemical or mechanical disruption of larger 37-kDa pneumococcal surface adhesin protein. An immunoreactive fragment is defined as an amino acid sequence of at least about 6 consecutive amino acids derived from the naturally occurring amino acid sequence, which has the relevant activity, e.g., evoking an immune response.
The fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the immunoreactivity of the peptide is not significantly impaired compared to the 37-kDa pneumococcal surface adhesin protein. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, etc. In any case, the peptide must possess a bioactive property, such as immunoreactivity. Functional or active regions of the 37-kDa pneumococcal surface adhesin protein may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the receptor. (See, e.g., Smith, M. xe2x80x9cIn vitro mutagenesisxe2x80x9d Ann. Rev. Gen., 19:423-462 (1985) and Zoller, M. J. xe2x80x9cNew molecular biology methods for protein engineeringxe2x80x9d Curr. Opin. Struct. Biol., 1:605-610 (1991)).
Antibodies
The present invention also provides a purified antibody which selectively binds with the polypeptide encoded by the nucleic acid set forth in the sequence listing as SEQ ID NO:1, or a polypeptide encoded by a unique fragment of at least 10 nucleotides of SEQ ID NO:1. The antibody (either polyclonal or monoclonal) can be raised to the 37-kDa pneumococcal surface adhesin protein of a unique fragment thereof, in its naturally occurring form and in its recombinant form. The antibody can be used in techniques or procedures such as diagnostics, treatment, or vaccination.
Antibodies can be made by many well-known methods (See, e.g. Harlow and Lane, xe2x80x9cAntibodies; A Laboratory Manualxe2x80x9d Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988)). Briefly, purified antigen can be injected into an animal in an amount and in intervals sufficient to elicit an immune response. Antibodies can either be purified directly, or spleen cells can be obtained from the animal. The cells can then fused with an immortal cell line and screened for antibody secretion. The antibodies can be used to screen nucleic acid clone libraries for cells secreting the antigen. Those positive clones can then be sequenced. (See, for example, Kelly et al., Bio/Technology, 10:163-167 (1992); Bebbington et al., Bio/Technology, 10:169-175 (1992)).
The phrase xe2x80x9cselectively bindsxe2x80x9d with the polypeptide refers to a binding reaction which is determinative of the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bound to a particular protein do not bind in a significant amount to other proteins present in the sample. Selective binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats may be used to select antibodies selectively bind with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies selectively immunoreactive with a protein. See Harlow and Lane xe2x80x9cAntibodies, A Laboratory Manualxe2x80x9d Cold Spring Harbor Publications, New York, (1988), for a description of immunoassay formats and conditions that could be used to determine selective binding.
In some instances, it is desirable to prepare monoclonal antibodies from various hosts. A description of techniques for preparing such monoclonal antibodies may be found in Stites et al., editors, xe2x80x9cBasic and Clinical Immunology,xe2x80x9d (Lange Medical Publications, Los Altos, Calif., Fourth Edition) and references cited therein, and in Harlow and Lane (xe2x80x9cAntibodies, A Laboratory Manualxe2x80x9d Cold Spring Harbor Publications, New York, (1988)).
The present invention also provides a monoclonal antibody designated 1E7A3D7C2, or a fragment thereof which retains the characteristics of antibody 1E7A3D7C2, such as its binding specificity and its binding affinity.
The present invention also provides a monoclonal antibody designated 1B6E12H9, or a fragment thereof which retains the characteristics of antibody 1B6E12H9.
The present invention also provides a monoclonal antibody designated 3C4D5C7, or a fragment thereof which retains the characteristics of antibody 3C4D5C7.
The present invention also provides a monoclonal antibody designated 4E9G9D3, or a fragment thereof which retains the characteristics of antibody 4E9G9D3.
The present invention also provides a monoclonal antibody designated 4H5C10F3, or a fragment thereof which retains the characteristics of antibody 4H5C10F3.
The present invention also provides a monoclonal antibody designated 6F6F9C8, or a fragment thereof which retains the characteristics of antibody 6F6F9C8.
The present invention also provides a monoclonal antibody designated 8G12G11B10, or a fragment thereof which retains the characteristics of antibody 8G12G11B10.
Vaccines
Also provided by thy present invention is a vaccine comprising an immunogenic polypeptide encoded by the nucleic acid as set forth in the Sequence Listing as SEQ ID NO:1, or a unique fragment of at least 10 nucleotides of SEQ ID NO:1. The polypeptides provided by the present invention can be used to vaccinate a subject for protection from a particular disease, infection, or condition caused by the organism from which the 37-kDa pneumococcal surface adhesin protein of a unique fragment thereof was derived.
Polypeptides of a 37-kDa pneumococcal surface adhesin protein of serotype 6B or a unique fragment thereof therefore, can be used to inoculate a host organism such that the host generates an active immune response to the presence of the polypeptide or polypeptide fragment which can later protect the host from infection by organism from which the polypeptide was derived. One skilled in the art will appreciate that an immune response, especially a cell-mediated immune response, to a 37-kDa pneumococcal surface adhesin protein from a specific strain can provide later protection from reinfection or from infection from a closely related strain. The 37-kDa protein provided by the present invention, however, is relatively conserved among many of the various serotypes of S. pneumoniae and can serve as a multivalent vaccine.
Immunization with the 37-kDa pneumococcal surface adhesin protein can be achieved through artificial vaccination. (Kuby, J. xe2x80x9cImmunologyxe2x80x9d W. H. Freeman and Co. New York, 1992). This immunization may be achieved by administering to subjects the 37-kDa pneumococcal surface adhesin protein either alone or with a pharmaceutically acceptable carrier.
Immunogenic amounts of the 37-kDa pneumococcal surface adhesin protein can be determined using standard procedures. Briefly, various concentrations of the present polypeptide are prepared, administered to subjects, and the immunogenic response (e.g., the production of antibodies to the polypeptide or cell mediated immunity) to each concentration is determined. Techniques for monitoring the immunogenic response, both cellular and humoral, of patients after inoculation with the polypeptide, are very well known in the art. For example, samples can be assayed using enzyme-linked immunosorbent assays (ELISA) to detect the presence of specific antibodies, such as serum IgA (Hjelt et al. J. Med. Virol. 21:39-47, (1987)), or lymphocyte or cytokine production can be monitored. The specificity of a putative immunogenic antigen of any particular polypeptide can be ascertained by testing sera, other fluids or lymphocytes from the inoculated patient for cross-reactivity with other closely related 37-kDa pneumococcal surface adhesin proteins.
The amount of a polypeptide of the 37-kDa pneumococcal surface adhesin protein administered will depend on the subject, the condition of the subject, the size of the subject, etc., but will be at least an immunogenic amount. The polypeptide can be formulated with adjuvants and with additional compounds, including cytokines, with a pharmaceutically acceptable carrier.
It is also contemplated that immunization against Streptococcus pneumoniae can be achieved by a xe2x80x9cnakedxe2x80x9d DNA vaccine approach. Briefly, DNA constructs containing promoter sequences upstream of the 37-kDa protein or specific antigen coding sequences can be injected into muscle tissue or administered via the mucosa and result in expression of viral antigens that induce a protective immune response.
The pharmaceutically acceptable carrier or adjuvant in the vaccine of the present invention can be selected by standard criteria (Arnon, R (Ed.) xe2x80x9cSynthetic Vaccinesxe2x80x9d I:83-92, CRC Press, Inc. Boca Raton, Fla., 1987). By xe2x80x9cpharmaceutically acceptablexe2x80x9d is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected compound without causing any undesirable biological effects or interacting in a undesirable manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier or adjuvant may depend on the method of administration and the particular patient.
Methods of administration can be by oral, sublingual mucosal, inhaled, absorbed, or by injection. Actual methods of preparing the appropriate dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington""s Pharmaceutical Sciences (Martin, E. W. (ed.) latest edition Mack Publishing Co., Easton, Pa.
Parenteral administration, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system, such that a constant level of dosage is maintained. See, e.g., U.S. Pat. No. 3,710,795, which is incorporated by reference herein.
Detection Methods
The present invention also provides a method of detecting the presence of the Streptococcus pneumoniae in a sample, comprising the steps of contacting a sample suspected of containing Streptococcus pneumoniae with nucleic acid primers capable of hybridizing to a nucleic acid comprising a unique portion of the nucleic acid set forth in the Sequencing Listing as SEQ ID NO:1, amplifying the nucleic acid, detecting the presence of an amplification product, the presence of the amplification product indicating the presence of Streptococcus pneumoniae in the sample. Alternatively, a unique fragment of the nucleic acid of SEQ ID NO:1 can be used to specifically identify a non-selectively amplified nucleic acid.
The specific amplification methods are well known in the art. For example, and as disclosed in the Example contained herein the polymerase chain reaction (PCR) can be used to amplify nucleic acid in a sample specific for Streptococcus pneumoniae. Other amplification techniques can also be used to detect the presence of Streptococcus pneumoniae in a sample, such as the ligase chain reaction (LCR), the self-sustained sequence replication (3SR) system, the transcription-based amplification system (TAS), and the RNA replication system based on Qxcex2 replicase.
The amplified nucleic acid can be detected in any number of detection assays. For example, the primers can be radio-labeled such that the amplification product containing these primers can be detected by the detecting the radioactive decay from those primers. Alternatively, the primers can contain other detectable moieties, such as biotin, or the amplified nucleic acid can be stained and visualized, such as with ethidium bromide staining.
The present invention also provides a method of detecting the presence of Streptococcus pneumoniae in a subject, comprising the steps of contacting an antibody-containing sample from the subject with purified polypeptide encoded by the nucleic acid set forth in the Sequence Listing as SEQ ID NO:1, or a purified polypeptide encoded by a nucleic acid comprising a unique fragment of at least 10 nucleotides of SEQ ID NO:1, and detecting the binding of the antibody with the polypeptide, the binding indicating the presence of Streptococcus pneumoniae in the subject.
The present invention further provides a method of detecting the presence of Streptococcus pneumoniae in a subject, comprising the steps of contacting a sample from the subject with an antibody which selectively binds the purified polypeptide encoded by the nucleic acid set forth in the Sequence Listing as SEQ ID NO:1, or a purified polypeptide encoded by a nucleic acid comprising a unique fragment of at least 10 nucleotides of SEQ ID NO:1 and detecting the binding of the antibody with an antigen, the binding indicating the presence of Streptococcus pneumoniae in the subject.
There are numerous immunodiagnostic methods that can be used to detect antigen or antibody as the following non-inclusive examples illustrate. These methods, as well as others, can not only detect the presence of antigen or antibody, but quantitate antigen or antibody as well.
Immunoassays such as immunofluorescence assays (IFA), enzyme linked immunosorbent assays (ELISA) and immunoblotting can be readily adapted to accomplish the detection and quantitation of the antigen or antibody. An ELISA method effective for the detection of the antigen, for example, can be as follows: (1) bind the antibody to a substrate; (2) contact the bound antibody with a fluid or tissue sample containing the antigen; (3) contact the above with a secondary antibody bound to a detectable moiety (e.g., horseradish peroxidase enzyme or alkaline phosphatase enzyme); (4) contact the above with the substrate for the enzyme; (5) contact the above with a color reagent; (6) observe color change. The above method can be readily modified to detect antibody as well as antigen.
Another immunologic technique that can be useful in the detection utilizes monoclonal antibodies (MAbs) for detection of antibodies that specifically bind a specific antigen. Briefly, sera or other body fluid from the subject is reacted with the antigen bound to a substrate (e.g. an ELISA 96-well plate). Excess sera is thoroughly washed away. A labeled (enzyme-linked, fluorescent, radioactive, etc.) monoclonal antibody is then reacted with the previously reacted antigen-serum antibody complex. The amount of inhibition of monoclonal antibody binding is measured relative to a control (no patient serum antibody). The degree of monoclonal antibody inhibition can be a specific test for a particular species or subspecies or variety or strain since it is based on monoclonal antibody binding specificity. MAbs can also be used for detection directly in cells by IFA.
A micro-agglutination test can also be used to detect the presence of antibodies in a subject. Briefly, latex beads (or red blood cells) are coated with the antigen and mixed with a sample from the subject, such that antibodies in the tissue or body fluids that are specifically reactive with the antigen crosslink with the antigen, causing agglutination. The agglutinated antigen-antibody complexes form a precipitate, visible with the naked eye or detectable by a spectrophotometer. In a modification of the above test, antibodies specifically reactive with the antigen can be bound to the beads and antigen in the tissue or body fluid thereby detected.
In addition, as in a typical sandwich assay, the antibody can be bound to a substrate and contacted with the antigen. Thereafter, a labeled secondary antibody is bound to epitopes not recognized by the first antibody and the secondary antibody is detected.
In the diagnostic methods taught herein, the antigen can be bound to a substrate and contacted by a fluid sample such as serum, urine, saliva or gastric juice. This sample can be taken directly from the subject, or in a partially purified form. In this manner, antibodies specific for the antigen (the primary antibody) will specifically bind with the bound antigen. Thereafter, a secondary antibody bound to, or labeled with, a detectable moiety can be added to enhance the detection of the primary antibody. Generally, the secondary antibody or other ligand which binds specifically with a different epitope of the antigen or nonspecifically with the ligand or bound antibody, will be selected for its ability to bind with multiple sites on the primary antibody. Thus, for example, several molecules of the secondary antibody can bind with each primary antibody, making the primary antibody more detectable.
The detectable moiety will allow visual detection of a precipitate or a color change, visual detection by microscopy, or automated detection by spectrometry, radiometric measurement or the like. Examples of detectable moieties include fluorescein and rhodamine (for fluorescence microscopy), horseradish peroxidase (for either light or electron microscopy and biochemical detection), biotin-streptavidin (for light or electron microscopy) and alkaline phosphatase (for biochemical detection by color change). The detection methods and moieties used can be selected, for example, from the list above or other suitable examples by the standard criteria applied to such selections (Harlow et al., xe2x80x9cAntibodies: A Laboratory Manualxe2x80x9d Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988)).
Methods of Treating and Preventing Infection
The present invention also provides a method of preventing Streptococcus pneumoniae infection in a subject, comprising administering to the subject a prophylactically effective amount of a vaccine comprising an immunogenic polypeptide encoded by the nucleic acid encoding the 37-kDa protein of Streptococcus pneumoniae as set forth in the Sequence Listing as SEQ I) NO:1, or an immunogenic polypeptide encoded by a nucleic acid comprising a unique fragment of at least 10 nucleotides of SEQ ID NO:1, either alone or with a pharmaceutically acceptable carrier.
The present invention further provides a method of preventing Streptococcus pneumoniae infection in a subject, comprising administering to the subject a prophylactically effective amount of an anti-idiotype antibody to the polypeptide encoded by the nucleic acid as set forth in the Sequence Listing as SEQ ID NO:1, or a polypeptide encoded by a nucleic acid comprising a unique fragment of at least 10 nucleotides of SEQ ID NO:1, either alone or with a pharmaceutically acceptable carrier.
Anti-idiotype antibodies represent the image of the original antigen and can serve as a vaccine to induce an immune response to a pathogenic antigen, therefore avoiding immunization with the pathogen itself This type of protection has been demonstrated by immunizing mice with anti-idiotype antibody to the binding site of TEPC-15, the major component of the pneumococcal cell wall C polysaccharide. Mice immunized with these anti-idiotype antibodies were immune when they were later challenged with live pneumococci. Mice have also been used to demonstrate anti-idiotype antibodies can provide protection against hepatitis B virus, rabies virus, Sendai virus, Streptococcus pneumoniae, Listeria monocytogenes, Trypanosoma rhodesiense, and Schistosoma mansoni. (See, Kuby, J. xe2x80x9cImmunologyxe2x80x9d W. H. Freeman and Co. New York, 1992).
The present invention further provides a method of treating a Streptococcus pneumoniae infection in a subject, comprising administering to the subject a therapeutically effective amount of an antibody to the polypeptide encoded by the nucleic acid as set forth in the Sequence Listing as SEQ ID NO:1, or a polypeptide encoded by a nucleic acid comprising a unique fragment of at least 10 nucleotides of SEQ ID NO:1, either alone or with a pharmaceutically acceptable carrier.
Treating a subject already infected with a particular organism by administering to the subject antibody against the organism is well known in the art. For example, immune globulin isolated from animals or humans previously exposed to rabies virus is currently a therapy for rabies virus infection. Better treatment of infected individuals can be achieved by administering to those individuals monoclonal antibodies since those monoclonals react or bind more specifically that the polyclonals. (See, e.g. Kaplan et at. xe2x80x9cRabiesxe2x80x9d Sci. Am. 242:120-134 (1980)).
The following example is put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the attenuated prokaryotes claimed herein are made and evaluated, and demonstrates the methods of the present invention, and is intended to be purely exemplary of the invention and is not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in xc2x0 C. and pressure is at or near atmospheric.